Herpes simplex viruses types 1 and 2 (HSV) are common human viruses that establish lifelong latency in sensory ganglia and reactivate periodically to reinfect mucosal tissues. While many HSV infections are relatively benign, HSV infections of the cornea can have serious consequences. HSV ocular infections trigger inflammatory pathology in the corneal stroma, normally an immunoprivileged tissue, producing a disease known as herpes stromal keratitis (HSK). HSK frequently involves recurring infections, over months or years, produced by cycles of reactivation and anterograde spread from ganglia to the eye. Over time, inflammation in the cornea caused by repeated reinfections can produce significant scarring and blindness. There are 50,000 new and recurrent cases of HSK every year in the U.S. and HSV remains the leading infectious cause of blindness. HSV coevolved with its host, allowing millions of years in which the virus has studied the nervous system. In neuronal axons, HSV hitchhikes on motor proteins that move rapidly along microtubule highways from nerve cell bodies in ganglia toward axon tips in epithelium. This fast axonal transport is essential for HSV survival, to allow virus production in the face of fully primed host immunity, and spread to other hosts. Two HSV membrane proteins: gE/gI and US9 are involved in hijacking axon transport machinery. gE/gI and US9 will serve us as molecular handles to investigate the poorly understood mechanisms by which HSV is transported in neuronal axons. Aim 1 will investigate how HSV gE/gI and US9 promote axonal transport of viral structural components. We showed that an HSV mutant lacking both gE/gI and US9 was completely blocked in transport of viral glycoproteins and capsids into distal axons. Two mechanisms are proposed to explain how gE/gI and US9 promote axonal transport. The Loading mechanism suggests that gE/gI and US9 function in neuronal cell bodies to sort and load HSV structural proteins onto microtubule motors. The Adaptor mechanism suggests that gE/gI and US9 function in axons to tether viral proteins onto kinesin motors. Aim 2 will involve efforts to identfy sorting motifs in gE/gI and US9 that promote either loading or adaptor functions. We have mutant gE molecules that may allow us to distinguish between these two mechanisms. Aim 3 will investigate which kinesin motors are involved in HSV axonal transport by: i) imaging fluorescent cellular cargo molecules in axons to determine if these are colocalized with HSV proteins, ii) shRNA silencing of kinesins and iii) using a new approach involving split kinesins. Together these studies will substantially advance our understanding of how HSV navigates in neuronal axons and provide important new information that can be used to design better drugs or vaccines.